Members of the epidermal growth factor receptor (EGFR) and c-Src tyrosine kinase families are frequently co-overexpressed in human neoplasias. This co-overexpression suggests that members of the two families may functionally interact to affect tumor formation and/or progression. Results from recent studies in both a model fibroblast system and in human breast cancer cell lines and tumor tissues have indicated that c-Src and EGFR synergistically interact to promote tumor formation. Evidence also supports a functional interaction between c-Src and two other members of the EGFR family, HER2 and HER3. The potentiation of EGFR-mediated tumor formation by c-Src correlates with EGF-induced physical association of the EGFR with c-Src, c-Src-dependent phosphorylation of the receptor at two novel sites (Y845 and Yl101), and increased tyrosine phosphorylation of selected receptor substrates. Substitution of phenylalanine for Y845 ablates EGF- and serum-induced DNA synthesis, suggesting that phosphorylation of Y845 may be a critical regulator of EGF-mediated growth and may represent a novel target to be exploited for tumor therapy. However, whether this site is required for malignant transformation mediated by the EGFR is not known, nor is the mechanism of phospho-Y845 (pY845) action understood. Similarly, the mechanism of the interplay between HER2/3 and c-Src is unknown. To further investigate the dependency of EGF-induced breast cancer cell growth and tumorigenesis on pY845 and the nature of the HER2/3/c-Src interaction, the ability of an inducibly expressed mutant Y845F EGFR or microinjected peptides containing pY845 to inhibit breast cancer cell growth in culture or in nude mice will be directly tested, as will biological synergism between c-Src and EGFR and its dependency on Y845 phosphorylation in a transgenic mouse model. The downstream effectors of phosphorylated Y845 will also be identified from a panel of known EGFR substrates and from differential phage display libraries, using phosphorylated vs. unphosphorylated peptides containing Y845 as probes. Finally the influence of c-Src on HER2/3-promoted cell survival, growth, and migration will be explored in two cell culture/xenograft models that will allow one to investigate the interaction in a normal (not transformed) cell context. The long-term goal of these studies is to understand the biological consequences of c-Src/EGFR family member interactions and the biochemical mechanisms that underlie them, thereby identifying potential new targets for therapeutic intervention of breast and other cancers.